Effect of IL-1β on β-cell function: insulin secretion, apoptose and proliferation.

<p>(A) Insulin secretion assay on INS-1 cells cultured in the presence of increasing IL-1β concentrations for 2 days. At the end of the exposure time, cells were washed, deprived in glucose during 1 hour and incubated in the presence of 0, 2.8, 5.6, 8.3 and 16 mM glucose. Cells supernatant fractions were collected and insulin content extracted with acid/alcohol mixture. Insulin present in culture supernatants and insulin content was quantified using HTRF assay. (B) Insulin secretion with IL-1β treatment (White column), and after pre-treatment of INS-1 cells with 500 ng/ml of IL1 receptor 1 antagonist (IL1-RA) to inhibit IL1-β effect on secretion (Black column). Insulin has been quantified by HTRF. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102889#pone-0102889-g002" target="_blank">Fig (2A-2B</a>): All data represent insulin release normalized for insulin contents and are expressed as percentages of insulin secretion recorded in the presence of 16 mM glucose alone; they are all the result of 3 independent experiments, with each experimental condition performed in triplicate. (C) Annexin V labeling of apoptotic INS-1 cells after treatment by increased concentrations of IL-1β: Ins-1 cells were analyzed by confocal microscopy (pictures are representative of 3 independent experiments) and flow cytometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102889#pone-0102889-g003" target="_blank">Fig 3B</a>). (D) Cell proliferation test by BrdU incorporation in INS-1 cells after IL-1β treatment; controls are cells not exposed to the cytokine. Data represent means of 3 independent experiments with each experimental condition performed in triplicates. *: p<0.05 and **: p<0.01 vs cells incubated in the absence of IL-1 β; a: p<0.05 and b: p<0.01 vs in the absence of antagonist.</p

Lipid rafts integrity and IL-1β effects on insulin secretion, apoptosis and cell proliferation.

<p>(A) Insulin secretion after MβCD treatment to induce cholesterol depletion and disturb lipid raft domains. INS-1 cells were stimulated by increasing concentrations of IL1-β and insulin secretion was quantified by HTRF assay. INS-1 cell without MβCD treatment (White column), and INS-1 cell pre-treated by MβCD (Black column). All data represent insulin release normalized for insulin contents and are expressed as percentages of insulin secretion recorded in the absence of any treatment. (B) INS-1 apoptosis. INS-1 cells were stimulated by IL-1β with or without MβCD pre-treatment, and induction of apoptosis was determined using Annexin V FITC/propidium iodide staining in flow cytometry. (C) Analysis of INS-1 proliferation using BrdU incorporation, with or without MβCD treatment and after IL-1β stimulation. INS-1 cell without MβCD treatment (White column), and INS-1 cell pre-treated by MβCD (Black column). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102889#pone-0102889-g003" target="_blank">Fig 3</a> (A-B-C) Data are means of 3 experiments with each experimental condition performed as triplicates. *: p<0.05 and **: p<0.01 vs non- IL-1β pretreated cells; a: p<0.05 and b: p<0.01 vs non- MβCD treated cells.</p

INS-1 cell line expresses IL-1R1 in lipid rafts.

<p>(A) Immunofluorescence studies in the INS-1 cell line; Immunostaining of receptor IL1-R1 and cytokine IL1-β. Irrelevant rabbit IgG and secondary antibody were used as negative controls. (B) Characterization of lipid raft fractions by Ganglioside M1 detection after isolation on sucrose gradient; Dot Blot characterization of Brij 98-extracted fractions. INS-1 cells were lysed with Brij 98 detergent at 37°C and separated into fractions by sucrose density gradient. Membrane rafts are present in fractions 3 and 4, as indicated by GM1 expression. Membrane lipid rafts localization in the INS-1 cell line by GM1 detection using immunofluorescence, without and with MβCD treatment. Results are representative of three independent experiments. (C) Western blots of IL-1R1 expression in lipid-rafts and non-rafts fractions. Forty micrograms of protein extracts from membrane lipid rafts- and non-lipid rafts were run on SDS-PAGE and blotted with anti Il-1R1 antibody.</p

IL1-β effects on protein expression in lipid raft- and non-lipid raft fractions.

<p>Changes in the expression pattern of proteins involved in signal transduction, apoptosis, and cell cycle were examined by antibody Array. Protein extracts (1 mg/ml) from INS-1 cells membrane lipid rafts and non lipid rafts, treated or not by IL1-β, were labeled with Cy3 and Cy5. Equal amounts of labelled proteins were incubated with antibodies doted on nitrocellulose-coated glass slides. The level of protein expression was quantified and the most representative changes are summarized on this Table.</p><p>Up or down regulated membrane lipid raft and non lipid raft proteins after IL-1β treatment are presented in relative values recorded for the same proteins expressed in raft and non raft fraction in control condition (no IL-1β treatment). A 2-fold increase or decrease (bold) taken as cut-off value (positive or negative) is considered as significant.</p

Differential islet proteins expression.

<p>A) Protein expression detected by Antibody Array technique. Changes in the expression pattern of proteins involved in signal transduction, apoptosis, and cell cycle were examined by Ab Array. Equal amounts of labelled proteins extracted from <i>fa/fa</i> and <i>fa/+</i> isolated islets were incubated on nitrocellulose-coated glass slides. Up or down regulated <i>fa/fa</i> islet proteins were presented in percentage relative to values recorded for <i>fa/+</i> islet proteins. A 20% cut-off value (positive or negative) is considered as significant. B) Immunoblot confirmation of Ab array results. Forty micrograms of total <i>fa/fa</i> or <i>fa/+</i> islet extracts were run on SDS polyacrylamide gel and blotted. Blots were incubated with Grb2, Caspase 1, Erk1/2, Erk1/2 phosphospecific, JNK, JNK phosphospecific, IKKγ, cleaved Caspase-3, Bcl-10 and Bcl-x antibodies that displayed interesting differential expression in Ab array experiments. Experiments were all duplicated with highly reproducible data. Results were quantified by densitometry as percentage of ß-actin, they are plotted as percents relative to <i>fa/+</i> value taken as 0.</p

Cytokines and receptors expression on pancreatic sections from <i>fa/fa</i> and <i>fa/+</i> rats.

<p>Pancreatic sections from <i>fa/fa</i> and <i>fa/+</i> rats were immunostained with anti-insulin and anti-cytokines or cytokine receptors antibodies (anti-TNF-R1, -TNFα, -IL-6R, -IL6, -IL-1R1, -IL-1ß, -IFNγ-R, and –IFNγ). Merge images give the double staining with anti-insulin antibody in green and anti-cytokines or cytokine receptors antibodies in red.</p

Reduced insulin secretion in M19-deficient INS-1 cells.

<p>(<b>A</b>) Northern blot analysis of the <i>M19</i> gene in human tissues. Pa: pancreas; Ki: kidney; Sk: skeletal muscle; Li: liver; Lu: lung; Pl: placenta; Br: brain; He: heart. Molecular markers are shown on the left. (<b>B</b>) Fluorescence microscopy of INS-1 cells double-labeled with the M19-specific P70612 antibody (M19, merge; green) and the MitoTracker dye (MitoTracker, merge; red). (<b>C</b>) Cell fractionation of INS-1 cells. Proteins of the total cell lysate (Lys), the cytosolic (Cyt) and the mitochondria (Mi) fractions were subjected to Western immunobloting. The cytosolic protein tubulin, the mitochondrial protein VDAC and M19 are detected. (<b>D</b>) INS-1 cells were transfected with a control pHYPER vector (sh control) or with the pHYPER vector encoding a M19-specific shRNA (sh M19). Western immunoblot analysis of the cell extracts shows expression levels of M19 and the control protein, tubulin. ATP production was determined in these cells (<b>E</b>), and insulin secretion was measured under basal glucose conditions (<b>F</b>). Results are the mean ± SEM of five (<b>E</b>), or four (<b>F</b>) independent experiments. (*) indicates statistical significance at p<0.05.</p

Expression and localization of M19 in muscle cells.

<p>(<b>A</b>) Coomassie-blue stained gel and Western blot analysis of M19 in extracts from C2C12 cells grown in proliferation medium (d0) or placed in differentiation-promoting conditions for 2 to 9 days (d2 to d9). M19 is detected by the rabbit polyclonal P70612 antibody. (<b>B</b>, <b>C</b>) C2C12 myoblasts were grown in proliferation medium or (<b>D</b>, <b>E</b>) were placed in differentiation medium for 6 days, and then were double-labeled with the specific P70612 antibody (<b>B</b>, <b>D; green</b>) and an anti-cytochrome c antibody (<b>C</b>, <b>E; red</b>). There is a co-localization between the 2 detected proteins in C2C12 myoblasts (<b>B</b>, <b>C, merge</b>) and myotubes (<b>D</b>, <b>E, merge</b>) as indicated by arrowheads. (<b>F</b>, <b>G</b>) Double-label indirect immunofluorescence of mouse <i>Tibialis anterior</i> sections showing M19 (<b>F; green</b>) and cytochrome c (<b>G; red</b>). (<b>H</b>) After C2C12 cell fractionation, proteins from the cytosolic and the mitochondria fractions were separated by SDS-PAGE. Tubulin, COX IV and M19 are detected by Western immunobloting. (<b>I</b>) Purified mitochondria are subjected to limited degradation using increasing concentration of trypsin, from 0 to 80 µg/ml. The mitochondria are then lysed in Laemmli buffer. Tom40 and M19 are detected by Western immunobloting. (<b>J</b>) Purified mitochondria are disrupted with freeze/thaw cycles, followed by Na₂CO₃ precipitation. After centrifugation, the membrane fraction (memb) and the matrix/intermembrane space fraction (matrix) are analyzed by Western immunobloting using a VDAC antibody and the P70612 antibody.</p

Identification of a mitochondrial targeting signal.

<p>(<b>A</b>) Prediction of the secondary structure of mouse M19 (<i>Mus musculus</i> NM026063) using 4 different algorithms: phyre, PSIPRED, SAM and jufo. The predicted α-helices are indicated by black lines along the amino-acid sequence. (<b>B</b>) Helical wheel presentation of the N-terminal α-helix of mouse M19, from amino acid 1 to 13. Hydrophobic residues are indicated in black circles while the positively charged amino acids are mentioned with a “+”. The first methionine (amino acid 1), at the top of the figure, is considered as a positively charged residue. (<b>C</b>, <b>D</b>) C2C12 myoblasts were transfected with the pQETriSystem vector encoding histidine-tagged M19 (<b>C</b>) or a histidine-tagged M19 mutant lacking amino acids 1 to 12 (<b>D</b>). Indirect immunofluorescence was performed using an anti-histidine antibody. (<b>E</b>–<b>J</b>) C2C12 myoblasts were transfected with the pEGFP-N1 vector encoding GFP alone (<b>E</b>, <b>F</b>), the pEGFP-N1 vector encoding the N-terminal M19 α-helix fused to the N-terminal end of GFP (<b>G</b>, <b>H</b>), and the PEGFP-C3 vector encoding the N-terminal M19 α-helix coupled to the C-terminal end of GFP (<b>I</b>, <b>J</b>). Fluorescence microscopy allows the direct detection of GFP constructs (<b>E</b>, <b>G</b>, <b>I; green</b>) and the indirect detection of cytochrome c using an anti-cytochrome c antibody (<b>F</b>, <b>H</b>, <b>J; red</b>).</p

M19 expression levels regulate mitochondrial ATP production in HeLa cells.

<p>(<b>A</b>, <b>B</b>) HeLa cells were transfected with a control siRNA (si control), a human M19-specific siRNA (si M19), a control siRNA associated with the pEGFP-N1 vector encoding mouse M19 (si control/M19GFP), or a human M19-specific siRNA associated with the pEGFP-N1 vector encoding mouse M19 (si M19/M19GFP). (<b>A</b>) Expression levels of the endogenous human M19 and the mouse GFP-coupled M19 are detected by Western immunobloting using the P70612 antibody. (<b>B</b>) ATP production is presented for these transfected-HeLa cells. Results are the mean ± SEM of five independent experiments. (***) indicates statistical significance at p<0.001, according to the unpaired Student's <i>t</i> test. (<b>C</b>) HeLa cells were transfected with a control siRNA (si control) or a human M19-specific siRNA (si M19). ATP production was then determined in untreated or in oligomycin-treated cells (oligo). Results are the mean ± SEM of four independent experiments. (**) indicates statistical significance at p<0.01, and (n.s.) means statistically non significant, according to the Tukey HSD test used after performing a one-way analysis of variance.</p